The following background discussion includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
In preclinical testing and clinical diagnostics, in vitro cellular assays are often used to test compound efficacy, toxicity, and host of other measurable outcomes related the in vivo pathological condition. For high-throughput lead compound validation experiments in preclinical screening, 96-well, 384-well, or 1536-well microtiter plates are employed to study cellular-compound interaction. Often these studies use a flat, glass or plastic (e.g., polystyrene) substrates to which cells adhere and grow in a monolayer-called two-dimensional (2D) cell culture. Such 2D cell culture techniques have demonstrated inconsistencies with the actual in vivo outcome, prompting the fields of engineering, biology, and medicine collectively to develop novel techniques to study cells at a more complex, three-dimensional (3D) physiologically relevant environment, called 3D cell culture. See e.g., W. Asghar, R. El Assal, H. Shafiee, S. Pitteri, R. Paulmurugan, U. Demirci, Engineering cancer microenvironments for in vitro 3D tumor models, Materials Today, 2015, 18 (10): 539-553; E. Knight, S. Przy Borski, Advances in 3D Cell Culture Technologies enabling tissue-like structures to be created in vitro, Journal of Anatomy, 2015, 227 (6): 746-756.
Some progress has been made using scaffold-free and scaffold-based approaches to 3D cell culture. In scaffold-free methodologies, cells aggregate into aggregates, which may have specific 3D shapes and placement. For example, multicellular sphere-like aggregates, or spheroids, form using hanging-drop and magnetic levitation methods. Spheroids also form in cultures grown in containers, such as round-bottom wells, V- and U-shaped wells on microtiter plates, and ultra-low attachment surfaces. See e.g., W. Asghar et al., supra; PCT/US2015/050522; U.S. Pat. No. 9,267,103B2; US20120171744A1; US20140322806A1.
However, scaffold-free techniques suffer from numerous disadvantages. First, scaffold-free techniques limit the user to studying spheroid growth and viability/toxicity in bulk measurements. Second, scaffold-free techniques do not take into account important aspects of tumor progression such as invasion, metastasis, and angiogenesis in the context of the tumor microenvironment (e.g., stromal cells, vascular cells, macrophages, extracellular matrix).
Scaffold-based techniques build 3D multi-cellular structures for 3D assays from extracellular matrixes, biomaterials, and/or polymeric structures. In the resulting structures, cells collectively respond to matrix, paracrine, and cellular cues to induce invasion, matrix degradation, or differentiation. Scaffold materials include natural, animal-based materials (e.g., basement membrane extract, or Matrigel; collagen), plant-based materials (e.g., alginate), synthetic (e.g., poly-ethylene glycol), or natural-synthetic combination and derivative materials (e.g., methacrylated hyaluronic acid or methacrylic gelatin). See e.g., W. Asghar et al., supra; S. Caliari, J. A. Burdick, A practical guide to hydrogels for cell culture, Nature Methods, 2016, 13 (5): 405-414; E. Jabbari, “Three dimensional matrix for cancer stem cells”, patent application Ser. No. 14/527,028, October 2014.
Synthetic and synthetic-natural hybrid polymeric biomaterials offer considerable advantages in modular material properties, as well as control over growth factor and binding site presentation and concentration. Modified dextran (e.g., dextran methacylate), gelatin (e.g., gelatin methacrylamide and/or gelatin methacrylate), hyaluronic acid (e.g., hyaluronic acid methacrylate), polyvinyl alcohol (e.g., acrylic acid modified PVA, acrylamide modified PVA) and polyethylene glycol (e.g., polyethylene glycol diacrylate), their derivatives, and combinations, have proven particularly useful in understanding disease progression in 3D cellular models. See e.g., K. Nguyen, J. L. West, Photopolymerizable hydrogels for tissue engineering applications, Biomaterials, 2002, 23: 4307-4314; S. Pedron, A. C. Harley, Impact of the biophysical features of a 3D gelatin microenvironment on glioblastoma malignancy, Journal of Biomedical Research A, 2013, 101(12): 3404-3415; B. Ananthanarayanan, Y. Kim, S. Kumar, Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform, Biomaterials, 2011, 32: 7913-7923. However, there remains a need for apparatuses and methods that quickly and reproducibly fabricate such materials into tissue-mimetic cell culture scaffolds. In other words, apparatuses and processes that form cell culture scaffolds that have properties similar to the in vivo cellular environment by patterning structures (e.g., venous structures) and controlling stiffness, density, porosity, adhesion properties, water content, pH, composition, heat and mass transport etc.
Recently, researchers have used photo-crosslinkable biomaterials for studying cancer progression. Typically, a light-sensitive prepolymer solution is formulated and mixed with cells and polymerized using UV light. A pattern within the matrix may be introduced via a photomask placed between the light source and the material, such that only the light passing through the mask design will crosslink the light-sensitive matrix below. Typically, cancer cells are mixed with the prepolymer solution so that they can be encapsulated within the matrix. See e.g., N. Peela, F. S. Sam, W. Christenson, D. Truong, A. W. Watson, G. Mouneimme, R. Ros, M. Nikkah, A three dimensional micropatterned tumor model for breast cancer cell migration studies, Biomaterials, 2016, 81: 72-83. Although this approach is simple, the resulting matrices fail to provide an environment in which cells migrate as if they were in living tissue, Rather, the individual cells may either adhere to one another, to the matrix, or escape the matrix and migrate out of the gel randomly. This random cell response leads to lower reproducibility and complicates measurements of overall tumor growth and invasion.
A second hurdle in current 3D cancer cell culture is enabling long time points for tumor spheroid growth and invasion. Hanging drop studies are practical for only tumor growth and viability studies, without any ability to study tumor cell invasion into extracellular matrix. Spheroid invasion assays are typically performed in Matrigel. However, due to Matrigel's soft (<1 kPa) and easily cell degradable matrix, experiments may not last beyond several days. Cellular processes such as invasion, angiogenesis, and transdifferentation (whereby tumor cells differentiate into other cell types) may be take weeks. One research group incorporated a tumor spheroid into a polymerized matrix that was wedged between two molds. However, invasion and quantifiable growth were not observed, which may be due to the experimental setup and lack of nutrient transport to the tumor. See e.g., A. Aung, J. Theprungsirikul, H. L. Lim, S. Varghese, Chemotaxis-driven assembly of endothelial barrier in a tumor-on-a-chip platform, Lab on a chip, 2016, 16: 1886-1898. Thus, a platform having improved durability and mass (e.g., nutrient, oxygen, carbon dioxide, waste etc.) transport is needed for studying and assaying controlled invasion and tumor growth over long time points.
A third hurdle for cellular assay analysis is studying 3D tumor cell culture in a high-throughput manner (e.g., multi-well plates) without the possibility of culture disruption during liquid media changes (removal via aspiration and re-application via pipetting) and for easy imaging. Currently in both spheroid-only and gel-based assays, sample loss or disruption to the microenvironment is likely to occur, diminishing reproducibility. It is highly necessary to limit any external (e.g., human) errors during media exchange in 3D cell culture.
To address the technical challenges posed by these hurdles, hydrogels have been patterned by extrusion bio-printing into each well, but again using a well-by-well process. These processes are laborious in nature when having to scale into multi-well plates for higher-throughput assays. Cell viability issues arise when cells remain in pre-polymer solution for long durations. And an extrusion process that uses a fine tip causes shear stress that can damage or kill cells, and is limited to using only soft, shearing materials, which are not representative of the tumor microenvironment. It would be beneficial to pattern gels into all wells at once, with the flexibility of material stiffness, to enhance scalability, reproducibility, and customization of the hydrogel structures and increase viability of the cells within and on the gels.
Some progress has been made by forming hydrogels using digital light projection (DLP), stereolithography (SLA), or general photo-patterning, between a glass slide and coverslip, and the resulting gel on glass is added to each well of a multi-well plate. In DLP, the projected image, or mask, is limited by the projector optics, which can cause image clarity and light uniformity issues across the area of the mask.
For example, WO 2015/179572 to Chung et al. discloses a system for 3D microfabrication that projects light toward a light modulator that modulates light responsive to digital masks corresponding to layers of a structure. A series of images corresponding to the digital masks are projected on a photopolymerizable material while a stage controls the optical plane. However, the system requires sophisticated equipment, and fabrication in multi-well plates can only be done using multiple systems or rapidly scanning across multiple wells.
Nikkhah et al. disclose a comparatively simple system in Patent Pub. No. US 2017/0067025. Nikkhah creates a high stiffness construct by cross-linking a first solution that includes cancer cells within a spacer. A second solution is disposed around the high stiffness constructs and crosslinked. Migration of cancer cells from the high stiffness construct to the low stiffness construct can then be observed. However, Nikkhah does not appreciate (1) that hydrogel-based tumor models can be rapidly fabricated within multi-well plates in parallel using a mold to control the height of the hydrogels and (2) that a space between the sides of the hydrogels and the well walls facilitates nutrient transport and exchange of liquid media. See also U.S. Pat. No. 8,906,684 to Bhatia et al.
These and all other extrinsic materials discussed herein are incorporated by reference in their entirety. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.
It has yet to be appreciated that organ models (e.g., tumor models comprising tumor and non-tumor cells in hydrogels having stiffnesses that mimic tumor and healthy tissue, respectively) may be easily prepared. Thus, there is still a need for apparatuses and processes for making organ, tissue, and tumor models that exhibit more accurate drug responses, especially in high-throughput diagnostics and therapeutic assays.